The adult skeleton is a dynamic system. Bone is continuously being formed and resorbed, starting in the embryo and continuing throughout adult life. This remodeling of bone is accomplished by precise coordination between two cell types: osteoblasts, which deposit the calcified bone matrix; and osteoclasts, which resorb bone. The sequence is always the same - activation of osteoclast precursors and osteoclastic bone resorption, followed by osteoblastic bone formation to repair the defects. Therefore, migration of osteoprogenitors to the resorptive site in response to osteoclastic bone resorption is an initial and central step in the coupling mechanism. However, the cellular and hormonal mechanisms responsible for mediating the coupling process are still not known. We have demonstrated that bone resorption-conditioned medium induces migration of MSCs. Neutralizing anti-TGF-b1 antibodies and depletion of TGF-b1 from the conditioned medium almost completely inhibit the migration whereas neutralizing antibodies to other potential coupling factors do not have such a dramatic effect. Conditioned medium from culture of osteoclasts with bone contains active TGF-b1, whereas conditioned medium from culture of osteoclasts or their precursors in the absence of bone contains the latent inactive form of TGF-b1 only. In characterization of the mechanisms involved in TGF-b1-induced cell migration, we have identified a novel RhoA signaling pathway that enhances cell adhesion and a Smad- dependent pathway that drives cell migration. The effects of inhibiters of RhoA and Smad signaling were tested in vivo by injection in rat tibia. Injection of the RhoA inhibitor, which promotes migration in our in vitro assays, stimulated bone formation in vivo, whereas injection of an inhibitor of Smad signaling, which inhibits cell migration in vitro, decreased bone formation in vivo. Taken together, our in vitro and in vivo data indicate that TGF-b1 induces migration of MSCs in response to bone resorption. Therefore, we hypothesize that TGF-b1, released and activated during osteoclastic bone resorption, induces migration of osteoprogenitors to bone resportive site as the primary coupling factor. We will 1) confirm that TGF-b1 released during osteoclastic bone resorption induces migration of MSCs with the use of TGF-b1 knockout mice and CED TGF-b1 mutants; 2) characterize the mechanism of TGF-b-induced migration of MSCs; 3) examine the mechanisms of TGF-b1-induced migration of osteoprogenitors in vivo by injection of inhibitors for Rho A and Smad phosphorylation in rat tibia and analyzing CED TGF-b1 mutant transgenic mice. Ultimately, this study will lead to reveal a mechanism of coupling bone resorption and formation and improve the treatment for bone diseases with coupling disorder.